An immunological examination method using an antigen-antibody reaction has been widely applied in various clinical examinations. A typical procedure thereof is to bring a specimen such as blood, urine, sputum, saliva, and nasal discharge or a specimen-diluted solution thereof into contact with a labeled-antibody solution in which antibody is labeled with a coloring identification material such as an enzyme, noble metal colloid, stained latex, and pigment to allow the antigen in the specimen to specifically react with the labeled antibody to make an antigen-antibody immune complex, followed by determining the amount of the immune complex through visual observation or as an optical change to conduct a qualitative or quantitative determination of antigen in the specimen.
Various simplified diagnostic kits adapting the above examination method have been developed and commercially available as tools for simply and quickly carrying out examination and diagnosis with respect to virus infection, pregnancy judgment, identification of cancer disease. As an example thereof, the current situation of a simplified diagnostic kit for examination and diagnosis for influenza virus infection will be described.
Reagents and tools for simply and quickly diagnosing influenza virus have been rapidly put to practical use because therapeutically effective anti-virus pharmaceutical preparations have become applicable recently in addition to avoiding needless administration of the conventional antibiotics and analgesics. Therefore, simplified diagnostic kits based on ELISA (enzyme-linked immunosorbent assay) have been developed and available from various pharmaceutical companies. One of their examples is disclosed in JP-A-2001-124775 and JP-A-2000-230931. Since medicines effective to influenza A virus and influenza B virus, respectively, have appeared, test reagents and tools capable of identifying and discriminating the types A or B have been developed. As the representative example, the usage of a simplified diagnostic kit for the influenza infection which uses a colloidal gold labeled antibody solution will be described as follows. (Reference Example 1)
<Usage of Simplified Diagnostic Kit for Influenza Infection Using Conventional Colloidal Gold Labeled Antibody Solution (Sandwich Type Simplified Measurement Method Using Flow-Through Technique)>
A. At first, a specimen which is collected as nasal discharge or a wipe liquid from the nasal cavity is added to a dilute solution prepared by adding a surfactant to a buffer solution, followed by stirring. The resulting mixture, as a diluted liquid specimen (a pre-treated specimen solution), is filled in a first tubular container with the end thereof covered with a cap having a built-in filter.
B. The diluted liquid specimen is transferred from the first tubular container to a second tubular container through the filter of the cap, and several droplets of a suspension of colloidal gold labeled anti-influenza antibody (a colloidal gold labeled antibody solution) are then dropped into the second container, followed by mixing them well to form a complex of the influenza virus in the specimen and the colloidal gold labeled anti-influenza antibody. The second tubular container is provided with a filtered cap different from the one used in the above step A and then left to stand for 2 to 5 minutes.
C. The whole amount of the mixture in the second tubular container is injected into a test device (a device for immunoassay) filled with a carrier (a porous film material such as a membrane) on which anti-influenza antibody is immobilized and an absorbent (such as absorbent cotton or absorbent paper) to allow the carrier to adsorb the complex of the influenza virus in the specimen and the colloidal gold labeled anti-influenza antibody, and a washing solution is then injected into the test device to wash the carrier.
D. Subsequently, a reaction occurred on the test device is observed to carry out identification and diagnosis to determine influenza virus infection and the type (A or B) of the influenza virus.
The conventional simplified diagnostic kit for inspecting influenza infection is a simple examination tool, but with several disadvantages which should be improved.
More specifically, for carrying out the reaction of the diluted liquid specimen with the colloidal gold labeled antibody, an operator should drop the colloidal gold labeled antibody solution into the diluted liquid specimen as described above, so that complicated operations such as transfer from the first container to second container can be required. In addition, the amounts of the droplets of the colloidal gold labeled antibody solution tend to vary due to differences among individuals of operators, so that there is a difficulty in reproducibility of test results. Besides, the colloidal gold labeled antibody solution tends to aggregate alone depending on the temperature thereof, so that it may not pass through the filter cap. In addition, the colloidal gold labeled antibody is not washed and filtered, and may remain on the test device during the examination. Thus, the colloidal gold labeled antibody may color the whole carrier or be non-specifically bound to the antibody immobilized on the carrier, causing false coloration which tends to be a cause of being judged false positive. Particularly, in the case of under the low-temperature conditions or of a lean solution, the long-term storage more than one year will be speculated difficult.
As can be found in the above exemplified diagnostic kit for influenza virus infection, many of simplified diagnostic kits commercially available in the market, on which immunological examination methods is applied, adopt the process of mixing a diluted liquid specimen with a labeled antibody solution and forming an immunocomplex. Therefore, the points that should be improved in the diagnostic kits for inspecting influenza virus infection may directly correspond to those of the general simplified diagnostic kits commercially available in the market.
More specifically, in order to make a specimen and a labeled antibody react in the conventional diagnostic kits on the market, it is necessary to mix the diluted liquid specimen with a labeled antibody solution. In many cases, a method that an operator drops several droplets of the labeled antibody solution to the diluted liquid specimen has been adopted. In this method, however, the amounts of the droplets of the labeled antibody solution tend to vary due to differences among individuals of operators. For instance, comparing between the cases of dropping one droplet and three droplets, the amounts of the solutions dropped will be much different from each other. If the amount of the labeled antibody solution dropped is small, a sufficient reaction cannot occur because of insufficient sensitivity. If the amount of the solution dropped is large, in the examination results errors due to the amount of droplet of the labeled antibody easily occur because of an increase in probability of causing a non-specific reaction or the like. Besides, there is a possibility of being difficult in correct measurement if the labeled antibody is stored for a long period in liquid form as it is. Therefore, the present invention tends to solve all of the problems of the conventional examination methods and tools.
In addition, the description in “Influenza Diagnostic Manual” edited by Minoru Kanazawa and Norio Sugaya (first printing, published on Feb. 20, 2001, Nankodo, Co., Ltd. pages 110-115) of a non-patent document will serve as a reference.